Methods for encapsulating plasmids in lipid bilayers

ABSTRACT

Plasmid-lipid particles which are useful for transfection of cells in vitro or in vivo are described. The particles can be formed using either detergent dialysis methods or methods which utilize organic solvents. The particles are typically 65-85 nm, fully encapsulate the plasmid and are serum-stable.

FIELD OF THE INVENTION

This invention relates to formulations for therapeutic nucleic acid delivery and methods for their preparation, and in particular to lipidencapsulated plasmids or antisense constructs. The invention provides acirculation-stable, characterizable delivery vehicle for theintroduction of plasmids or antisense compounds into cells. Thesevehicles are safe, stable, and practical for clinical use.

BACKGROUND OF THE INVENTION

Gene therapy is an area of current interest which involves theintroduction of genetic material into a cell to facilitate expression ofa deficient protein. There are currently five major methods by whichthis is accomplished, namely: (i) calcium phosphate precipitation, (ii)DEAE-dextran complexes, (iii) electroporation, (iv) cationic lipidcomplexes and (v) reconstituted viruses or virosomes (see Chang, et al.,Focus 10:88 (1988)). Cationic lipid complexes are presently the mosteffective generally used means of effecting transfection.

A number of different formulations incorporating cationic lipids arecommercially available, namely (i) LIPOFECTIN® (which uses1,2-dioleyloxy-3-(N,N,N-trimethylamino)propane chloride, or DOTMA, seeEppstein, et al., U.S. Pat. No. 4,897,355); LIPOFECTAMINE® (which usesDOSPA, see Hawley-Nelson, et al., Focus 15(3):73 (1993)); andLIPOFECTACE® (which uses N,N-distearyl-N,N-dimethyl-ammonium bromide, orDDAB, see Rose, U.S. Pat. No. 5,279,833). Others have reportedalternative cationic lipids that work in essentially the same manner butwith different efficiencies, for example1,2-dioleoyloxy-3-(N,N,N-trimethylamino)propane chloride, or DOTAP, seeStomatatos, et al., Biochemistry 27:3917-3925 (1988)); glycerol basedlipids (see Leventis, et al., Biochem. Biophys. Acta 1023:124 (1990);lipopolyamines (see, Behr, et al., U.S. Pat. No. 5,171,678) andcholesterol based lipids (see Epand, et al., WO 93105162, and U.S. Pat.No. 5,283,185).

Others have noted that DOTMA and related compounds are significantlymore active in transfection assays than their saturated analogues (see,Felgner, et al., WO91/16024). However, both DOTMA and DOSPA basedformulations, despite being the most efficient of the cationic lipids ineffecting transfection, are prohibitively expensive. DDAB on the otherhand is inexpensive and readily available from chemical suppliers but isless effective than DOTMA in most cell lines. Another disadvantage ofthe current lipid systems is that they are not appropriate forintravenous injection.

An examination of the relationship between the chemical structure of thecarrier vehicle and its efficiency of transfection has indicated thatthe characteristics which provide for effective transfection would makea carrier unstable in circulation (see, Ballas, et al., Biochim.Biophys. Acta 939:8-18 (1988)). Additionally, degradation either outsideor inside the target cell remains a problem (see, Duzghines, SubcellularBiochemistry 11:195-286 (1985)). Others who have attempted toencapsulate DNA (Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980);and Deamer, U.S. Pat. No. 4,515,736) made no efforts to ensure a safe,injectable formulation, or arrived at inefficient loading (Legendre,Pharm. Res. 9:1235-1242 (1992)).

Ideally, a delivery vehicle for a nucleic acid or plasmid will have thefollowing characteristics: a) small enough and long lived enough todistribute from local injection sites when given intravenously, b)capable of carrying a large amount of DNA per particle to enabletransfection of all sizes of genes and reduce the volume of injection,c) homogenous, d) reproducible, e) protective of DNA from extracellulardegradation and f) capable of transfecting target cells in such a waythat the DNA is not digested intracellularly.

The present invention provides such compositions and methods for theirpreparation and use.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides methods for thepreparation of serum-stable plasmid-lipid particles. In one group ofthese methods, a plasmid is combined with cationic lipids in a detergentsolution to provide a coated plasmid-lipid complex. The complex is thencontacted with non-cationic lipids to provide a solution of detergent, aplasmid-lipid complex and non-cationic lipids, and the detergent is thenremoved to provide a solution of serum-stable plasmid-lipid particles,in which the plasmid is encapsulated in a lipid bilayer. The particles,thus formed, have a size of about 50-150 nm.

In a related group of methods the serum-stable plasmid-lipid particlesare formed by preparing a mixture of cationic lipids and non-cationiclipids in an organic solvent; contacting an aqueous solution of plasmidwith the mixture of cationic and non-cationic lipids to provide a clearsingle phase; and removing the organic solvent to provide a suspensionof plasmid-lipid particles, in which the plasmid is encapsulated in alipid bilayer, and the particles are stable in serum and have a size ofabout 50-150 nm.

In another aspect, the present invention provides plasmid-lipidparticles prepared by the above methods.

In yet another aspect the present invention provides methods oftransfecting cells using these plasmid-lipid particles.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a liposome-mediated transfection using“sandwich-type” complexes of DNA.

FIG. 2 illustrates an aggregation and precipitation which commonlyoccurs during the entrapment of large nucleic acids in lipid complexes.

FIG. 3 provides a schematic representation of the preparation ofplasmid-lipid particles using the methods of the present invention.

FIG. 4 illustrates the recovery of ³H-DNA from encapsulated particlesfollowing the reverse-phase preparation of the particles and extrusionthrough a 400 nm filter and a 200 nm filter. Lipid composition isPOPC:DODAC:PEG-Cer(C₂₀) in proportions as shown in Table 1.

FIG. 5 illustrates the recovery of ³H-DNA from particles prepared usinga reverse-phase procedure. The particles were extruded through a 200 nmfilter and eluted on a DEAE Sepharose CL-6B anion exchange column. Thepercent recovery reported is based on the amount recovered afterfiltration. Lipid composition is as in FIG. 4.

FIG. 6 illustrates the recovery of ¹⁴C-lipid from encapsulated particlesfollowing the reverse-phase preparation of the particles and extrusionthrough a 400 nm filter and a 200 nm filter. Lipid composition is as inFIG. 4.

FIG. 7 illustrates the recovery of ¹⁴C-lipid from particles preparedusing a reverse-phase procedure. The particles were extruded through a200 nm filter and eluted on a DEAE Sepharose CL-6B anion exchangecolumn. The percent recovery reported is based on the amount recoveredafter filtration. Lipid composition is as in FIG. 4.

FIG. 8 illustrates recovery of ³H-DNA and ¹⁴C-lipids from particlesprepared by detergent dialysis after elution on a DEAE Sepharose CL-6Banion exchange column in HBS, pH 7.4. Lipid composition isPOPC:DODAC:PEG-Cer(C₂₀) in proportions as shown in Table 2.

FIG. 9 illustrates recovery of ³H-DNA and ¹⁴C-lipids from particlesprepared by detergent dialysis after elution on a DEAE Sepharose CL-6Banion exchange column in HBS, pH 7.4. Lipid composition isDOPE:DODAC:PEG-Cer(C₂₀) in proportions as shown in Table 3.

FIG. 10 provides an elution profile of free ³H-DNA (pCMV4-CAT) on aSepharose CL-4B column in HBS, pH 7.4.

FIG. 11 provides an elution profile of free ³H-DNA (pCMV4-CAT) on aSepharose CL-4B column in HBS, pH 7.4, after incubation in 80% mouseserum for 30 min at 37° C.

FIG. 12 shows the recovery of ³H-DNA and ¹⁴C-lipids from particles(prepared by reverse-phase methods) after incubation in 80% mouse serumfor 15 min at 37° C. Lipid composition is POPC:DODAC:PEG-Cer(C₂₀).

FIG. 13 shows the recovery of ³H-DNA and ¹⁴C-lipids from particles(prepared by detergent dialysis methods) after incubation in 80% mouseserum for 30 min at 37° C. Lipid composition is DOPE:DODAC:PEG-Cer(C₂₋₉.

FIG. 14 provides a density gradient profile of ¹⁴C-lipid complexesprepared in the absence of DNA by reverse phase methods. Lipidcomposition is POPC:DODAC:PEG-Cer(C₂₀).

FIG. 15 provides a density gradient profile of free ³H-DNA (pCMV4-CAT).

FIG. 16 provides a density gradient profile of ³H-DNA and ¹⁴C-lipid fromparticles prepared by reverse-phase methods. Lipid composition is as inFIG. 14.

FIG. 17 provides a density gradient profile of free ³H-DNA, ¹⁴C-lipidcomplexes prepared in the absence of DNA by detergent dialysis methodsand ³H-DNA and ¹⁴C-lipid from DNA-lipid complexes prepared by detergentdialysis. Lipid composition is DOPE:DODAC:PEG-Cer(C₂₀).

FIG. 18 provide a size distribution of DNA-lipid particles prepared bydetergent dialysis methods. Lipid composition isDOPE:DODAC:PEG-Cer(C₂₀).

FIG. 19 shows the clearance of ³H-DNA and ¹⁴C-lipid from particles(prepared by reverse-phase methods) after injection into IRC mice. Thefigure includes free ³H-DNA after injection as a comparison. Lipidcomposition is POPC:DODAC:PEG-Cer(C₂₀).

FIG. 20 shows the clearance of ³H-DNA and ¹⁴C-lipid from particles(prepared by detergent dialysis methods) after injection into IRC mice.Lipid composition is DOPE:DODAC:PEG-Cer(C₂₀) (83.5:6.5:10 mole %).

FIG. 21 shows the clearance of ³H-DNA and ¹⁴C-lipid from particles(prepared by detergent dialysis methods) after injection into IRC mice.Lipid composition is as in FIG. 20 except that PEG-Cer(C₂₀) is replacedwith PEG-Cer(C₁₄).

FIG. 22 shows the results of in vivo transfection which occurs in thelungs of mice. Lipid composition is DOPE:DODAC:PEG-Cer(C₂₀ or C₁₄)(83.5:6.5:10 mole %).

FIG. 23 shows the results of in vivo transfection which occurs in theliver of mice. Lipid composition is as in FIG. 22.

FIG. 24 shows the results of in vivo transfection which occurs in thespleen of mice. Lipid composition is as in FIG. 22.

DETAILED DESCRIPTION OF THE INVENTION Contents

I. Glossary

II. General

III. Methods of Forming Plasmid-Lipid Particles

IV. Pharmaceutical Preparations

V. Administration of Plasmid-Lipid Particle Formulations

VI. Examples

VII. Conclusion

I. GLOSSARY

The following abbreviations are used herein: DC-Chol,3β-(N—(N′,N′-dimethylaminoethane)carbamoyl)cholesterol (see, Gao, etal., Biochem. Biophys. Res. Comm. 179:280-285 (1991)); DDAB,N,N-distearyl-N,N-dimethylammonium bromide; DMRIE,N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide; DODAC, N,N-dioleyl-N,N-dimethylammonium chloride (see commonlyowned patent application U.S. Ser. No. 08/316,399, incorporated hereinby reference); DOGS, diheptadecylamidoglycyl spermidine; DOPE,1,2-sn-ioleoylphoshatidyethanolamine; DOSPA,N-1-(2,3-dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammoniumtrifluoroacetate; DOTAP,N1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammoniumchloride; DOTMA,N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride; EPC, eggphosphatidylcholine; RT, room temperature; HEPES,4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HBS, HEPES bufferedsaline (150 mM NaCl and 20 mM HEPES); PEG-Cer-C₂₀,1-O-(2′-(ω-methoxypolyethyleneglycol)succinoyl)-2-N-arachidoyl-sphingosine;PEG-Cer-C₁₄,1-O-(2′-(ω-methoxypolyethyleneglycol)succinoyl)-2-N-myristoyl-sphingosine;PBS, phosphate-buffered saline; EGTA,ethylenebis(oxyethylenenitrilo)-tetraacetic acid; OGP, n-octylβ-D-glycopyranoside (Sigma Chemical Co., St. Louis, Mo.); POPC,palmitoyl oleoyl phosphatidylcholine (Northern Lipids, Vancouver, BC);QELS, quasielastic light scattering; TBE, 89 mM Tris-borate with 2 mMEDTA; and EDTA, Ethylenediaminetetraacetic acid (Fisher Scientific, FairLawn, N.J.);

The term “acyl” refers to a radical produced from an organic acid byremoval of the hydroxyl group. Examples of acyl radicals include acetyl,pentanoyl, palmitoyl, stearoyl, myristoyl, caproyl and oleoyl.

The term “lipid” refers to any fatty acid derivative which is capable offorming a bilayer such that a hydrophobic portion of the lipid materialorients toward the bilayer while a hydrophilic portion orients towardthe aqueous phase. Hydrophilic characteristics derive from the presenceof phosphato, carboxylic, sulfato, amino, sulfhydryl, nitro, and otherlike groups. Hydrophobicity could be conferred by the inclusion ofgroups that include, but are not limited to, long chain saturated andunsaturated aliphatic hydrocarbon groups and such groups substituted byone or more aromatic, cycloaliphatic or heterocyclic group(s). Preferredlipids are phosphoglycerides and sphingolipids, representative examplesof which include phosphatidylcholine, phosphatidylethanolamine,phosphatidylserine, phosphatidylinositol, phosphatidic acid,palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine,lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine,dioleoylphosphatidylcholine, distearoylphosphatidylcholine ordilinoleoylphosphatidylcholine could be used. Other compounds lacking inphosphorus, such as sphingolipid and glycosphingolipid families are alsowithin the group designated as lipid. Additionally, the amphipathiclipids described above may be mixed with other lipids includingtriglycerides and sterols.

The term “non-cationic lipid” refers to any of a number of lipid specieswhich exist either in an uncharged form, a neutral zwitterionic form, oran anionic form at physiological pH. Such lipids include, for examplediacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide,sphingomyelin, cephalin, cardiolipin, and cerebrosides.

The term “cationic lipid” refers to any of a number of lipid specieswhich carry a net positive charge at physiological pH. Such lipidsinclude, but are not limited to, DODAC, DOTMA, DDAB, DOTAP, DC-Chol andDMRIE. Additionally, a number of commercial preparations of cationiclipids are available which can be used in the present invention. Theseinclude, for example, LIPOFECTIN® (commercially available cationicliposomes comprising DOTMA and DOPE, from GIBCO/BRL, Grand Island, N.Y.,USA); LIPOFECTAMINE® (commercially available cationic liposomescomprising DOSPA and DOPE, from GIBCO/BRL); and TRANSFECTAM®(commercially available cationic liposomes comprising DOGS from PromegaCorp., Madison, Wis., USA).

The terms “transfection” and “transformation” are used hereininterchangeably, and refer to the introduction of polyanionic materials,particularly nucleic acids, into cells. The term “lipofection” refers tothe introduction of such materials using liposome or lipid-basedcomplexes. The polyanionic materials can be in the form of DNA or RNAwhich is linked to expression vectors to facilitate gene expressionafter entry into the cell. Thus the polyanionic material used in thepresent invention is meant to include DNA having coding sequences forstructural proteins, receptors and hormones, as well as transcriptionaland translational regulatory elements (i.e., promoters, enhancers,terminators and signal sequences) and vectors. Methods of incorporatingparticular nucleic acids into expression vectors are well known to thoseof skill in the art, but are described in detail in, for example,Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed.), Vols.1-3, Cold Spring Harbor Laboratory, (1989) or Current Protocols inMolecular Biology, F. Ausubel et al., ed. Greene Publishing andWiley-Interscience, New York (1987), both of which are incorporatedherein by reference.

“Expression vectors”, “cloning vectors”, or “vectors” are often plasmidsor other nucleic acid molecules that are able to replicate in a chosenhost cell. Expression vectors may replicate autonomously, or they mayreplicate by being inserted into the genome of the host cell, by methodswell known in the art. Vectors that replicate autonomously will have anorigin of replication or autonomous replicating sequence (ARS) that isfunctional in the chosen host cell(s). Often, it is desirable for avector to be usable in more than one host cell, e.g., in E. coli forcloning and construction, and in a mammalian cell for expression.

II. GENERAL

Although directed to the transfer of nucleic acids, and in particular tothe transfer of plasmids to cells, the particles of the presentinvention can be used for delivering essentially any polyanionicmolecule. As noted in the Background of the Invention, typicallipid-nucleic acid formulations are formed by combining the nucleic acidwith a preformed cationic liposome (see, U.S. Pat. Nos. 4,897,355,5,264,618, 5,279,833 and 5,283,185. In such methods, the nucleic acid isattracted to the cationic surface charge of the liposome and theresulting complexes are thought to be of the “sandwich-type” depicted inFIG. 1. As a result, a portion of the nucleic acid or plasmid remainsexposed in serum and can be degraded by enzymes such as DNAse I. Othershave attempted to incorporate the nucleic acid or plasmid into theinterior of a liposome during formation. These methods typically resultin the aggregation in solution of the cationic lipid-nucleic acidcomplexes (see FIG. 2). Passive loading of a plasmid into a preformedliposome has also not proven successful. Finally, the liposome-plasmidcomplexes which have been formed are typically 200 to 400 nm in size andare therefore cleared more rapidly from circulation than smaller sizedcomplexes or particles. The present invention provides a method ofpreparing serum-stable plasmid-lipid particles in which the plasmid isencapsulated, n a lipid-bilayer and is protected from degradation.Additionally, the particles formed have a size of about 50 to about 150nm, with a majority of the particles being about 65 to 85 nm. Theparticles can be formed by either a detergent dialysis method or by amodification of a reverse-phase method which utilizes organic solventsto provide a single phase during mixing of the components. Withoutintending to be bound by any particular mechanism of formation, FIG. 3depicts a detergent dialysis approach to the formation of theplasmid-lipid particles. With reference to FIG. 3, a plasmid or otherlarge nucleic acid is contacted with a detergent solution of cationiclipids to form a coated plasmid complex. These coated plasmids canaggregate and precipitate. However, the presence of a detergent reducesthis aggregation and allows the coated plasmids to react with excesslipids (typically, non-cationic lipids) to form particles in which theplasmid is encapsulated in a lipid bilayer. As noted above, theseparticles differ from the more classical liposomes both in size(liposomes being typically 200400 nm) in that there is little or noaqueous medium encapsulated by the particle's lipid bilayer. The methodsdescribed below for the formation of plasmid-lipid particles usingorganic solvents follow a similar scheme.

III. METHODS OF FORMING PLASMID-LIPID PARTICLES

The present invention provides methods for the formation of serum-stableplasmid-lipid particles. While the invention is described with referenceto the use of plasmids, one of skill in the art will understand that themethods described herein are equally applicable to other larger nucleicacids or oligonucleotides. In one group of embodiments, the particlesare formed using detergent dialysis. Thus, the present inventionprovides a method for the preparation of serum-stable plasmid-lipidparticles, comprising:

-   -   (a) combining a plasmid with cationic lipids in a detergent        solution to form a coated plasmid-lipid complex;    -   (b) contacting non-cationic lipids with the coated plasmid-lipid        complex to form a detergent solution comprising a plasmid-lipid        complex and non-cationic lipids; and    -   (c) dialyzing the detergent solution of step (b) to provide a        solution of serum-stable plasmid-lipid particles, wherein the        plasmid is encapsulated in a lipid bilayer and the particles are        serum-stable and have a size of from about 50 to about 150 nm.

The plasmids which are useful in the present invention are typicallynucleotide polymers which are to be administered to a subject for thepurpose of repairing or enhancing the expression of a cellular protein.Accordingly, the nucleotide polymers can be polymers of nucleic acidsincluding genomic DNA, cDNA, or mRNA. Still further, the plasmids mayencode promoter regions, operator regions, structural regions. Whennucleic acids other than plasmids are used the nucleic acids can containnucleic acid analogs, for example, the antisense derivatives describedin a review by Stein, et al., Science 261:1004-1011 (1993) and in U.S.Pat. Nos. 5,264,423 and 5,276,019, the disclosures of which areincorporated herein by reference.

The plasmids, or nucleic acids can be single-stranded DNA or RNA, ordouble-stranded DNA or DNA-RNA hybrid. Examples of double-stranded DNAinclude structural genes, genes including operator control andtermination regions, and self-replicating systems such as plasmid DNA.

Single-stranded nucleic acids include antisense oligonucleotides(complementary to DNA and RNA), ribozymes and triplex-formingoligonucleotides. In order to have prolonged activity, thesingle-stranded nucleic acids will preferably have some or all of thenucleotide linkages substituted with stable, non-phosphodiesterlinkages, including, for example, phosphorothioate, phosphorodithioate,phosphoroselenate, or O-alkyl phosphotriester linkages.

The nucleic acids used in the present invention will also include thosenucleic acids in which modifications have been made in one or more sugarmoieties and/or in one or more of the pyrimidine or purine bases.Examples of sugar modifications include replacement of one or morehydroxyl groups with halogens, alkyl groups, amines, azido groups orfunctionalized as ethers or esters. Additionally, the entire sugar maybe replaced with sterically and electronically similar structures,including aza-sugars and carbocyclic sugar analogs. Modifications in thepurine or pyrimidine base moiety include, for example, alkylated purinesand pyrimidines, acylated purines or pyrimidines, or other heterocyclicsubstitutes known to those of skill in the art.

Multiple genetic sequences can be also be used in the present methods.Thus, the sequences for different proteins may be located on one strandor plasmid. Promoter, enhancer, stress or chemically-regulatedpromoters, antibiotic-sensitive or nutrient-sensitive regions, as wellas therapeutic protein encoding sequences, may be included as required.Non-encoding sequences may be also be present, to the extent they arenecessary to achieve appropriate expression.

The nucleic acids used in the present method can be isolated fromnatural sources, obtained from such sources as ATCC or GenBank librariesor prepared by synthetic methods. Synthetic nucleic acids can beprepared by a variety of solution or solid phase methods. Generally,solid phase synthesis is preferred. Detailed descriptions of theprocedures for solid phase synthesis of nucleic acids byphosphite-triester, phosphotriester, and H-phosphonate chemistries arewidely available. See, for example, Itakura, U.S. Pat. No. 4,401,796;Caruthers, et al., U.S. Pat. Nos. 4,458,066 and 4,500,707; Beaucage, etal., Tetrahedron Lett., 22:1859-1862 (1981); Matteucci; et al., J. Am.Chem. Soc., 103:3185-3191 (1981); Caruthers, et al., GeneticEngineering, 4:1-17 (1982); Jones, chapter 2, Atkinson, et al., chapter3, and Sproat, et al., chapter 4, in Oligonucleotide Synthesis: APractical Approach, Gait (ed.), IRL Press, Washington D.C. (1984);Froehler, et al., Tetrahedron Lett., 27:469-472 (1986); Froehler, etal., Nucleic Acids Res., 14:5399-5407 (1986); Sinha, et al. TetrahedronLett., 24:5843-5846 (1983); and Sinha, et al., Nucl. Acids Res.,12:4539-4557 (1984) which are incorporated herein by reference.

Cationic lipids which are useful in the present invention, include, forexample, DODAC, DOTMA, DDAB, DOTAP, DC-Chol and DMRIE. These lipids andrelated analogs, which are also useful in the present invention, havebeen described in co-pending U.S. Ser. No. 08/316,399; U.S. Pat. Nos.5,208,036, 5,264,618, 5,279,833 and 5,283,185, the disclosures of whichare incorporated herein by reference. Additionally, a number ofcommercial preparations of cationic lipids are available and can be usedin the present invention. These include, for example, LIPOFECTIN®(commercially available cationic liposomes comprising DOTMA and DOPE,from GIBCO/BRL, Grand Island, N.Y., USA); LIPOFECTAMINE® (commerciallyavailable cationic liposomes comprising DOSPA and DOPE, from GIBCO/BRL);and TRANSFECTAM® (commercially available cationic liposomes comprisingDOGS from Promega Corp., Madison, Wis., USA).

An initial solution of coated plasmid-lipid complexes is formed bycombining the plasmid with the cationic lipids in a detergent solution.The detergent solution is preferably an aqueous solution of a neutraldetergent having a critical micelle concentration of 15-300 mM, morepreferably 20-50 mM. Examples of suitable detergents include, forexample, N,N′-((octanoylimino)-bis-(trimethylene))-bis-(D-gluconamide)(BIGCHAP); BRIJ 35; Deoxy-BIGCHAP; dodecylpoly(ethylene glycol) ether;Tween 20; Tween 40; Tween 60; Tween 80; Tween 85; Mega 8; Mega 9;Zwittergent® 3-08; Zwittergent® 3-10; Triton X-405; hexyl-, heptyl-,octyl- and nonyl-β-D-glucopyranoside; and heptylthioglucopyranoside;with octyl β-D-glucopyranoside being the most preferred. Theconcentration of detergent in the detergent solution is typically about100 mM to about 2 M, preferably from about 200 mM to about 1.5 M.

The cationic lipids and plasmid will typically be combined to produce acharge ratio (+/−) of about 1:1 to about 20:1, preferably in a ratio ofabout 1:1 to about 12:1, and more preferably in a ratio of about 2:1 toabout 6:1. Additionally, the overall concentration of plasmid insolution will typically be from about 25 μg/mL to about 1 mg/mL,preferably from about 25 μg/mL to about 200 μg/mL, and more preferablyfrom about 50 μg/mL to about 100 μg/mL. The combination of plasmids andcationic lipids in detergent solution is kept, typically at roomtemperature, for a period of time which is sufficient for the coatedcomplexes to form. Alternatively, the plasmids and cationic lipids canbe combined in the detergent solution and warmed to temperatures of upto about 37° C. For plasmids which are particularly sensitive totemperature, the coated complexes can be formed at lower temperatures,typically down to about 4° C.

The detergent solution of the coated plasmid-lipid complexes is thencontacted with non-cationic lipids to provide a detergent solution ofplasmid-lipid complexes and non-cationic lipids. The non-cationic lipidswhich are useful in this step include, diacylphosphatidylcholine,diacylphosphaddylethanolamine, ceramide, to sphingomyelin, cephalin,cardiolipin, and cerebrosides. In preferred embodiments, thenon-cationic lipids are diacylphosphatidylcholine,diacylphosphatidylethanolamine, ceramide or sphingomyelin. The acylgroups in these lipids are preferably acyl groups derived from fattyacids having C₁₀-C₂₄ carbon chains. More preferably the acyl groups arelauroyl, myristoyl, palmitoyl, stearoyl or oleoyl. In particularlypreferred embodiments, the noncationic lipid will be1,2-sn-dioleoylphosphatidylethanolamine (DOPE), palmitoyl oleoylphosphatidylcholine (POPC) or egg phosphatidylcholine (EPC). In the mostpreferred embodiments, the plasmid-lipid particles will be fusogenicparticles with enhanced properties in vivo and the non-cationic lipidwill be DOPE. In other preferred embodiments, the non-cationic lipidswill further comprise polyethylene glycol-based polymers such as PEG2000, PEG 5000 and polyethylene glycol conjugated to ceramides, asdescribed in co-pending U.S. Ser. No. 08/316,429, incorporated herein byreference.

The amount of non-cationic lipid which is used in the present methods istypically about 2 to about 20 mg of total lipids to 50 μg of plasmid.Preferably the amount of total lipid is from about 5 to about 10 mg per50 μg of plasmid.

Following formation of the detergent solution of plasmid-lipid complexesand non-cationic lipids, the detergent is removed, preferably bydialysis. The removal of the detergent results in the formation of alipid-bilayer which surrounds the plasmid providing serum-stableplasmid-lipid particles which have a size of from about 50 nm to about150 nm. The particles thus formed do not aggregate and are optionallysized to achieve a uniform particle size.

The serum-stable plasmid-lipid particles can be sized by any of themethods available for sizing liposomes. The sizing may be conducted inorder to achieve a desired size range and relatively narrow distributionof particle sizes.

Several techniques are available for sizing the particles to a desiredsize. One sizing method, used for liposomes and equally applicable tothe present particles is described in U.S. Pat. No. 4,737,323,incorporated herein by reference. Sonicating a particle suspensioneither by bath or probe sonication produces a progressive size reductiondown to particles of less than about 50 nm in size. Homogenization isanother method which relies on shearing energy to fragment largerparticles into smaller ones. In a typical homogenization procedure,particles are recirculated through a standard emulsion homogenizer untilselected particle sizes, typically between about 60 and 80 nm, areobserved. In both methods, the particle size distribution can bemonitored by conventional laser-beam particle size discrimination, orQELS.

Extrusion of the particles through a small-pore polycarbonate membraneor an asymmetric ceramic membrane is also an effective method forreducing particle sizes to a relatively well-defined size distribution.Typically, the suspension is cycled through the membrane one or moretimes until the desired particle size distribution is achieved. Theparticles may be extruded through successively smaller-pore membranes,to achieve a gradual reduction in size.

In another group of embodiments, the present invention provides a methodfor the preparation of serum-stable plasmid-lipid particles, comprising;

-   -   (a) preparing a mixture comprising cationic lipids and        non-cationic lipids in an organic solvent;    -   (b) contacting an aqueous solution of nucleic acid with said        mixture in step (a) to provide a clear single phase; and    -   (c) removing said organic solvent to provide a suspension of        plasmid-lipid particles, wherein said plasmid is encapsulated in        a lipid bilayer, and said particles are stable in serum and have        a size of from about 50 to about 150 nm.

The plasmids (or nucleic acids), cationic lipids and non-cationic lipidswhich are useful in this group of embodiments are as described for thedetergent dialysis methods above.

The selection of an organic solvent will typically involve considerationof solvent polarity and the ease with which the solvent can be removedat the later stages of particle formation. The organic solvent, which isalso used as a solubilizing agent, is in an amount sufficient to providea clear single phase mixture of plasmid and lipids. Suitable solventsinclude chloroform, dichloromethane, diethylether, cyclohexane,cyclopentane, benzene, toluene, methanol, or other aliphatic alcoholssuch as propanol, isopropanol, butanol, tert-butanol, iso-butanol,pentanol and hexanol. Combinations of two or more solvents may also beused in the present invention.

Contacting the plasmid with the organic solution of cationic andnon-cationic lipids is accomplished by mixing together a first solutionof plasmid, which is typically an aqueous solution and a second organicsolution of the lipids. One of skill in the art will understand thatthis mixing can take place by any number of methods, for example bymechanical means such as by using vortex mixers.

After the plasmid has been contacted with the organic solution oflipids, the organic solvent is removed, thus forming an aqueoussuspension of serum-stable plasmid-lipid particles. The methods used toremove the organic solvent will typically involve evaporation at reducedpressures or blowing a stream of inert gas (e.g., nitrogen or argon)across the mixture.

The serum-stable plasmid-lipid particles thus formed will typically besized from about 50 nm to 150 nm. To achieve further size reduction orhomogeneity of size in the particles, sizing can be conducted asdescribed above.

In other embodiments, the methods will further comprise adding nonlipidpolycations which are useful to effect the transformation of cells usingthe present compositions. Examples of suitable nonlipid polycationsinclude, hexadimethrine bromide (sold under the brandname POLYBRENE®,from Aldrich Chemical Co., Milwaukee, Wis., USA) or other salts ofheaxadimethrine. Other suitable polycations include, for example, saltsof poly-L-ornithine, poly-L-arginine, poly-L-lysine, poly-D-lysine,polyallylamine and polyethyleneimine.

In other embodiments, the polyoxyethylene conjugates which are used inthe plasmid-lipid particles of the present invention can be prepared bycombining the conjugating group (i.e. phosphatidic acid orphosphatidylethanolamine) with an appropriately functionalizedpolyoxyethylene derivative. For example, phosphatidylethanolamine can becombined with polyoxyethylene bis(p-toluenesulfonate) to provide aphosphatidylethanolamine-polyoxyethylene conjugate. See, Woodle, et al.,Biochim. Biophys. Acta 1105:193-200 (1992), incorporated herein byreference.

The present invention also provides plasmid-lipid particles which areprepared by the methods described above. In preferred embodiments, theparticles comprise a plasmid, a non-cationic lipid which is a mixture ofPOPC and PEG-Cer or DOPE and PEG-Cer, and a cationic lipid which isDODAC.

IV. PHARMACEUTICAL PREPARATIONS

The plasmid-lipid particles of the present invention can be administeredeither alone or in mixture with a physiologically-acceptable carrier(such as physiological saline or phosphate buffer) selected inaccordance with the route of administration and standard pharmaceuticalpractice.

Pharmaceutical compositions comprising the plasmid-lipid particles ofthe invention are prepared according to standard techniques and furthercomprise a pharmaceutically acceptable carrier. Generally, normal salinewill be employed as the pharmaceutically acceptable carrier. Othersuitable carriers include, e.g., water, buffered water, 0.4% saline,0.3% glycine, and the like, including glycoproteins for enhancedstability, such as albumin, lipoprotein, globulin, etc. Thesecompositions may be sterilized by conventional, well known sterilizationtechniques. The resulting aqueous solutions may be packaged for use orfiltered under aseptic conditions and lyophiled, the lyophilizedpreparation being combined with a sterile aqueous solution prior toadministration. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiologicalconditions, such as pH adjusting and buffering agents, tonicityadjusting agents and the like, for example, sodium acetate, sodiumlactate, sodium chloride, potassium chloride, calcium chloride, etc.Additionally, the particle suspension may include lipid-protectiveagents which protect lipids against free-radical and lipid-peroxidativedamages on storage. Lipophilic free-radical quenchers, such asalphatocopherol and water-soluble iron-specific chelators, such asferrioxamine, are suitable.

The concentration of particles in the pharmaceutical formulations canvary widely, i.e., from less than about 0.05%, usually at or at leastabout 2-5% to as much as 10 to 30% by weight and will be selectedprimarily by fluid volumes, viscosities, etc., in accordance with theparticular mode of administration selected. For example, theconcentration may be increased to lower the fluid load associated withtreatment. This may be particularly desirable in patients havingatherosclerosis-associated congestive heart failure or severehypertension. Alternatively, particles composed of irritating lipids maybe diluted to low concentrations to lessen inflammation at the site ofadministration. For diagnosis, the amount of particles administered willdepend upon the particular label used, the disease state being diagnosedand the judgement of the clinician but will generally be between about0.01 and about 50 mg per kilogram of body weight, preferably betweenabout 0.1 and about 5 mg/kg of body weight.

As noted above, it is often desirable to include polyethylene glycol(PEG), PEG-ceramide, or ganglioside G_(Ml)-modified lipids to theparticles. Addition of such components prevents particle aggregation andprovides a means for increasing circulation lifetime and increasing thedelivery of the plasmid-lipid particles to the target tissues.Typically, the concentration of the PEG, PEG-ceramide orGM_(Ml)-modified lipids in the particle will be about 1-15%.

Overall particle charge is also an important determinant in particleclearance from the blood, with negatively charged complexes being takenup more rapidly by the reticuloendothelial system (Juliano, Biochem.Biophys. Res. Commun. 63:651 (1975)) and thus having shorter half-livesin the bloodstream. Particles with prolonged circulation half-lives aretypically desirable for therapeutic and diagnostic uses. For instance,particles which can be maintained from 8, 12, or up to 24 hours in thebloodstream are particularly preferred.

In another example of their use, plasmid-lipid particles can beincorporated into a broad range of topical dosage forms including butnot limited to gels, oils, emulsions and the like. For instance, thesuspension containing the plasmid-lipid particles can be formulated andadministered as topical creams, pastes, ointments, gels, lotions and thelike.

The present invention also provides plasmid-lipid particles in kit form.The kit will typically be comprised of a container which iscompartmentalized for holding the various elements of the kit. The kitwill contain the compositions of the present inventions, preferably indehydrated form, with instructions for their rehydration andadministration. In still other embodiments, the particles and/orcompositions comprising the particles will have a targeting moietyattached to the surface of the particle. Methods of attaching targetingmoieties (e.g., antibodies, proteins) to lipids (such as those used inthe present particles) are known to those of skill in the art.

Dosage for the plasmid-lipid particle formulation will depend on theratio of nucleic acid to lipid and the administrating physician'sopinion based on age, weight, and condition of the patient.

V. ADMINISTRATION OF PLASMID-LIPID PARTICLE FORMULATIONS

The serum-stable plasmid-lipid particles of the present invention areuseful for the introduction of plasmids into cells. Accordingly, thepresent invention also provides methods for introducing a plasmid into acell. The methods are carried out in vitro or in vivo by first formingthe particles as described above, then contacting the particles with thecells for a period of time sufficient for transfection to occur.

The particles of the present invention can be adsorbed to almost anycell type. Once adsorbed, the particles can either be endocytosed by aportion of the cells, exchange lipids with cell membranes, or fuse withthe cells. Transfer or incorporation of the nucleic acid portion of theparticle can take place via any one of these pathways. In particular,when fusion takes place, the particle membrane is integrated into thecell membrane and the contents of the particle combine with theintracellular fluid. Contact between the cells and the plasmid-lipidparticles, when carried out in vitro, will take place in a biologicallycompatible medium. The concentration of particles can vary widelydepending on the particular application, but is generally between about1 μmol and about 10 mmol. Treatment of the cells with the plasmid-lipidparticles will generally be carried out at physiological temperatures(about 37° C.) for periods of time of from about 1 to 6 hours,preferably of from about 2 to 4 hours. For in vitro applications, thedelivery of nucleic acids can be to any cell grown in culture, whetherof plant or animal origin, vertebrate or invertebrate, and of any tissueor type. In preferred embodiments, the cells will be animal cells, morepreferably mammalian cells, and most preferably human cells.

In one group of preferred embodiments, a plasmid-lipid particlesuspension is added to 60-80% confluent plated cells having a celldensity of from about 10³ to about 10⁵ cells/mL, more preferably about2×10⁴ cells/mL. The concentration of the suspension added to the cellsis preferably of from about 0.01 to 0.2 μg/mL, more preferably about 0.1μg/mL.

Typical applications include using well known transfection procedures toprovide intracellular delivery of DNA or mRNA sequences which code fortherapeutically useful polypeptides. However, the compositions can alsobe used for the delivery of the expressed gene product or proteinitself. In this manner, therapy is provided for genetic diseases bysupplying deficient or absent gene products (i.e., for Duchenne'sdystrophy, see Kunkel, et al., Brit. Med. Bull. 45(3):630-643 (1989),and for cystic fibrosis, see Goodfellow, Nature 341:102-103 (1989)).Other uses for the compositions of the present invention includeintroduction of antisense oligonucleotides in cells (see, Bennett, etal., Mol. Pharm. 41:1023-1033 (1992)).

Alternatively, the compositions of the present invention can also beused for the transfection of cells in vivo, using methods which areknown to those of skill in the art. In particular, Zhu, et al., Science261:209-211 (1993), incorporated herein by reference, describes theintravenous delivery of cytomegalovirus (CMV)-chloramphenicolacetyltransferase (CAT) expression plasmid using DOTMA-DOPE complexes.Hyde, et al., Nature 362:250-256 (1993), incorporated herein byreference, describes the delivery of the cystic fibrosis transmembraneconductance regulator (CFTR) gene to epithelia of the airway and toalveoli in the lung of mice, using liposomes. Brigham, et al., Am. J.Med. Sci. 298:278-281 (1989), incorporated herein by reference,describes the in vivo transfection of lungs of mice with a functioningprokaryotic gene encoding the intracellular enzyme, chloramphenicolacetyltransferase (CA).

For in vivo administration, the pharmaceutical compositions arepreferably administered parenterally, i.e., intraarticularly,intravenously, intraperitoneally, subcutaneously, or intramuscularly.More preferably, the pharmaceutical compositions are administeredintravenously or intraperitoneally by a bolus injection. For example,see Stadler, et al., U.S. Pat. No. 5,286,634, which is incorporatedherein by reference. Intracellular nucleic acid delivery has also beendiscussed in Straubringer, et al., METHODS IN ENZYMOLOGY, AcademicPress, New York. 101:512-527 (1983); Mannino, et al., Biotechniques6:682-690 (1988); Nicolau, et al., Crit. Rev. Ther. Drug Carrier Syst.6:239-271 (1989), and Behr, Acc. Chem. Res. 26:274-278 (1993). Stillother methods of administering lipid-based therapeutics are describedin, for example, Rahman et al., U.S. Pat. No. 3,993,754; Sears, U.S.Pat. No. 4,145,410; Papahadjopoulos et al., U.S. Pat. No. 4,235,871;Schneider, U.S. Pat. No. 4,224,179; Lenk et al., U.S. Pat. No.4,522,803; and Fountain et al., U.S. Pat. No. 4,588,578.

In other methods, the pharmaceutical preparations may be contacted withthe target tissue by direct application of the preparation to thetissue. The application may be made by topical, “open” or “closed”procedures. By “topical”, it is meant the direct application of thepharmaceutical preparation to a tissue exposed to the environment, suchas the skin, oropharynx, external auditory canal, and the like. “Open”procedures are those procedures which include incising the skin of apatient and directly visualizing the underlying tissue to which thepharmaceutical preparations are applied. This is generally accomplishedby a surgical procedure, such as a thoracotomy to access the lungs,abdominal laparotomy to access abdominal viscera, or other directsurgical approach to the target tissue. “Closed” procedures are invasiveprocedures in which the internal target tissues are not directlyvisualized, but accessed via inserting instruments through small woundsin the skin. For example, the preparations may be administered to theperitoneum by needle lavage. Likewise, the pharmaceutical preparationsmay be administered to the meninges or spinal cord by infusion during alumbar puncture followed by appropriate positioning of the patient ascommonly practiced for spinal anesthesia or metrazamide imaging of thespinal cord. Alternatively, the preparations may be administered throughendoscopic devices.

The plasmid-lipid particles can also be administered in an aerosolinhaled into the lungs (see, Brigham, et al., Am. J. Sci. 298(4):278-281(1989)) or by direct injection at the site of disease (Culver, HUMANGENE THERAPY, MaryAnn Liebert, Inc., Publishers, New York. pp. 70-71(1994)).

The methods of the present invention may be practiced in a variety ofhosts. Preferred hosts include mammalian species, such as humans,non-human primates, dogs, cats, cattle, horses, sheep, and the like.

VI. EXAMPLES

The following examples are offered solely for the purposes ofillustration, and are intended neither to limit nor to define theinvention. In each of these examples, the term “DNA” or “plasmid” refersto the plasmid pCMV4-CAT.

Example 1

This example illustrates the encapsulation of a plasmid in a lipidbilayer system using either a reverse-phase method or a detergentdialysis method.

Reverse Phase Method

pCMV4-CAT plasmid (50 μg) was encapsulated in a lipid bilayer which wasconstructed using 20 mg POPC:PEG-Cer-C₂₀ (95:5 mole % ratio) withbetween 0 and 0.3 mg DODAC. The encapsulation method utilized amodification of the classical reverse phase method for entrapment.Specifically, 1.050 mL of chloroform:methanol in a 1:2.1 mole % ratiowas added to a lipid film containing 2 μL of ¹⁴C-cholesteryl hexadecylether (6.66 μL/μCi). This was followed by the addition of 220 μL H₂O and33 μL ³H-pCMV4-CAT plasmid (158,000 dpm/μL; 1.5 mg/mL). This combinationprovided a clear single phase. The CHCl₃ and most of the methanol wereremoved under a stream of nitrogen while vortexing the mixture. Theresulting 250 μL suspension of encapsulated plasmid was diluted with 1mL of H₂O and extruded 5 times through one 400 nm filter followed byextrusion 5 times through one 200 nm filter. The resulting vesicle sizewas approximately 150 to 200 nm in diameter.

Detergent Dialysis Method

pCMVCAT (50 μg consisting of 20 μL of ³H-pCMV4-CAT and 30 μL of coldpCMV4-CAT at a concentration of 1 mg/mL, “plasmid”) was incubated withDODAC at various DODAC:plasmid charge ratios in 100 μL of 1Mn-octyl-β-D-glucopyranoside and 400 μL H₂O for 30 min at roomtemperature. The resulting plasmid:DODAC mixture was added to asuspension of 5 mg POPC:PEG-Cer(C₂₀) or 10 mg DOPE:PEG-Cer(C₂₀)(containing 1 μL ¹⁴C-cholesteryl hexadecyl ether; 6.66 μL/μCi) in 100 μLof 1 M n-octyl-β-D-glucopyranoside and 400 μL of H₂O). The amounts usedfor each lipid to achieve a desired charge ratio are shown in Tables1-3. The suspension was dialysed against HBS at pH 7.4 overnight. Theresulting encapsulated plasmid can be used without further sizing.

TABLE 1 Calculation of % DODAC in vesicles for any given DODAC:DNAcharge ratio as a function of total mg of lipid Lipid DNA POPC DODACPEGCer POPC DODAC PEGCer total lipid (mg) DODAC:DNA microgram % % % mgmg mg micromole 20 1 50 89.25979 0.740207 10 14.09342 0.0895 5.81707920.77528 20 2 50 88.52161 1.478393 10 13.99597 0.179 5.82503 20.80368 203 50 87.78543 2.214567 10 13.89852 0.2685 5.83298 20.83207 20 4 5087.05126 2.948737 10 13.80107 0.358 5.840931 20.86047 20 5 50 86.319093.68091 10 13.70362 0.4475 5.848882 20.88886 20 6 50 85.5889 4.411096 1013.60617 0.537 5.856832 20.91726 20 7 50 84.8607 5.139302 10 13.508720.6265 5.864783 20.94565 20 8 50 84.13446 5.865537 10 13.41127 0.7165.872734 20.97405 20 9 50 83.41019 6.589808 10 13.31382 0.8055 5.88068421.00244 20 10 50 82.68788 7.312122 10 13.21637 0.895 5.888635 21.0308420 11 50 81.96751 8.03249 10 13.11891 0.9845 5.896585 21.05923 20 12 5081.24908 8.750917 10 13.02146 1.074 5.904536 21.08763 20 13 50 80.532599.467412 10 12.92401 1.1635 5.912487 21.11602 20 14 50 79.81802 10.1819810 12.82656 1.253 5.920437 21.14442 20 15 50 79.10536 10.89464 1012.72911 1.3425 5.928388 21.17281 20 16 50 78.39462 11.60538 10 12.631661.432 5.936339 21.20121 20 17 50 77.68578 12.31422 10 12.53421 1.52155.944289 21.2296 20 18 50 76.97883 13.02117 10 12.43676 1.611 5.9522421.258 20 19 50 76.27376 13.72624 10 12.33931 1.7005 5.96019 21.28639 2020 50 75.57058 14.42942 10 12.24186 1.79 5.968141 21.31479

TABLE 2 Calculation of % DODAC in vesicles for any given DODAC:DNAcharge ratio as a function of total mg of lipid Lipid DNA POPC DODACPEGCer POPC DODAC PEGCer total lipid (mg) DODAC:DNA microgram % % % mgmg mg micromole 5 1 50 87.05126 2.948737 10 3.450267 0.0895 1.4602335.215117 5 2 50 84.13446 5.865537 10 3.352817 0.179 1.468183 5.243512 53 50 81.24908 8.750917 10 3.255366 0.2685 1.476134 5.271907 5 4 5078.39462 11.60538 10 3.157915 0.358 1.484085 5.300302 5 5 50 75.5705814.42942 10 3.060465 0.4475 1.492035 5.328697 5 6 50 72.77647 17.2235310 2.963014 0.537 1.499986 5.357092 5 7 50 70.01183 19.98817 10 2.8655630.6265 1.507937 5.385488 5 8 50 67.27619 22.72381 10 2.768113 0.7161.515887 5.413883 5 9 50 64.56909 25.43091 10 2.670662 0.8055 1.5238385.442278 5 10 50 61.8901 28.1099 10 2.573212 0.895 1.531788 5.470673 511 50 59.23877 30.76123 10 2.475761 0.9845 1.539739 5.499068 5 12 5056.61469 33.38531 10 2.37831 1.074 1.54769 5.527463 5 13 50 54.0174235.98258 10 2.28086 1.1635 1.55564 5.555858 5 14 50 51.44657 38.55343 102.183409 1.253 1.563591 5.584253 5 15 50 48.90173 41.09827 10 2.0859591.3425 1.571541 5.612648 5 16 50 46.38252 43.61748 10 1.988508 1.4321.579492 5.641043 5 17 50 43.88853 46.11147 10 1.891057 1.5215 1.5874435.669438 5 18 50 41.41941 48.58059 10 1.793607 1.611 1.595393 5.697833 519 50 38.97477 51.02523 10 1.696156 1.7005 1.603344 5.726228 5 20 5036.55426 53.44574 10 1.598705 1.79 1.611295 5.754623

TABLE 3 Calculation of % DODAC in vesicles for any given DODAC:DNAcharge ratio as a function of total mg of lipid Lipid DNA DOPE DODACPEGCer DOPE DODAC PEGCer total lipid (mg) DODAC:DNA microgram % % % mgmg mg micromole 10 1 50 88.54333 1.456667 10 6.954544 0.0895 2.95595610.55698 10 2 50 87.09389 2.906111 10 6.857699 0.179 2.963301 10.5832210 3 50 85.65161 4.348388 10 6.760853 0.2685 2.970647 10.60945 10 4 5084.21645 5.783549 10 6.664007 0.358 2.977993 10.63569 10 5 50 82.788357.211648 10 6.567162 0.4475 2.985338 10.66192 10 6 50 81.36726 8.63273610 6.470316 0.537 2.992684 10.68816 10 7 50 79.95314 10.04686 10 6.373470.6265 3.00003 10.71439 10 8 50 78.54591 11.45409 10 6.276625 0.7163.007375 10.74063 10 9 50 77.14555 12.85445 10 6.179779 0.8055 3.01472110.76686 10 10 50 75.752 14.248 10 6.082933 0.895 3.022067 10.7931 10 1150 74.3652 15.6348 10 5.986088 0.9845 3.029412 10.81933 10 12 5072.98511 17.01489 10 5.889242 1.074 3.036758 10.84556 10 13 50 71.6116818.38832 10 5.792396 1.1635 3.044104 10.8718 10 14 50 70.24487 19.7551310 5.69555 1.253 3.05145 10.89803 10 15 50 68.88462 21.11538 10 5.5987051.3425 3.058795 10.92427 10 16 50 67.53089 22.46911 10 5.501859 1.4323.066141 10.9505 10 17 50 66.18362 23.81638 10 5.405013 1.5215 3.07348710.97674 10 18 50 64.84279 25.15721 10 5.308168 1.611 3.080832 11.0029710 19 50 63.50833 26.49167 10 5.211322 1.7005 3.088178 11.02921 10 20 5062.1802 27.8198 10 5.114476 1.79 3.095524 11.05544

Example 2

This example illustrates the level of plasmid “protection” from externalmedium using anion exchange chromatography.

The extent of encapsulation or protection of the plasmid from theexternal medium was assessed by anion exchange chromatography asfollows: a 50 μL aliquot of each sample was eluted on a DEAE SepharoseCL-6B column and the fractions were assessed for both ³H-plasmid and¹⁴C-lipid by scintillation counting. Any exposed negative charges, suchas those present on DNA molecules will bind to the anion exchange columnand will not elute with the ¹⁴C-lipid. DNA which has its negative charge“protected” or non-exposed will not bind to the ion exchange resin andwill elute with the ¹⁴C-lipid.

Reverse Phase Method (Particles with POPC:DODAC:PEG-Cer(C₂₀)

FIG. 4 presents the results describing the relationship betweenDODAC:plasmid charge ratio (see Table 1 for amounts of POPC, DODAC andPEG-Cer(C₂₀) using 20 mg total lipid) and percent recovery of plasmidafter extrusion through a 400 nm filter and a 200 nm filter. An increasein percent plasmid recovered was observed corresponding to an increasein DODAC:plasmid charge ratio. No plasmid was recovered in the absenceof DODAC while, at a DODAC:plasmid charge ratio of 2:1, 90% of theplasmid was recovered after extrusion through a 400 nm filter and 70% ofthe plasmid was recovered after extrusion through a 200 nm filter.Nearly 100% of the plasmid recovered from extrusion through a 200 nmfilter was recovered by anion exchange chromatography (see FIG. 5)suggesting that all of the recovered plasmid was encapsulated. Thiscorresponded to an overall encapsulation efficiency of about 70%. Lipidrecoveries after extrusion and anion exchange chromatography were 90%after extrusion through a 400 nm filter and 70% after extrusion througha 200 nm filter (see FIG. 6). Of the 70% lipid recovered after extrusionthrough a 200 nm filter, nearly 100% was recovered after anion exchangechromatography (see FIG. 7). Lipid and plasmid recovery after extrusionand anion exchange chromatography were nearly identical.

Dialysis Method (Particles with POPC:DODAC:PEG-Cer(C₂₀)

FIG. 8 provides the results which illustrate the effect of DODAC:plasmidcharge ratio on the percent recovery of lipid and plasmid from anionexchange chromatography following preparation of the particles using thedetergent dialysis method of Example 1 (amounts of lipids are providedin Table 2 for 5 mg total lipid compositions). Significant protectionwas observed over a DODAC:plasmid charge ratio of about 3:1 to 5:1.Also, it appears that significant protection of the plasmid is achievedat a DODAC:plasmid charge ratio of about 8:1. The recovery of lipiddecreased from 100% in the absence of DODAC to about 85% at aDODAC:plasmid charge ratio of 8:1.

Dialysis Method (Particles with DOPE:DODAC:PEG-Cer(C₂₀)

In a similar manner to that described for the POPC-containing particles,the fusogenic lipid composition DOPE:DODAC:PEG-Cer(C₂₀) was assessed byanion exchange chromatography. Aliquots (50 μL) of the plasmid-lipidparticles (prepared by detergent dialysis, using the amounts provided inTable 3) were eluted on a DEAE Sepharose CL-6B column. FIG. 9 providesthe results and illustrates the relationship between the DODAC:DNAcharge ratio and % recovery of lipid and DNA for particles using 10 mgof total lipid. DNA encapsulation occurred at a DODAC:DNA charge ratioof 4:1.

Example 3

This example illustrates the serum stability achieved usingplasmid:lipid particles prepared by the methods of Example 1.

To establish the serum stability of the plasmid:lipid particles,aliquots of the particle mixtures prepared according to both the reversephase and dialysis methods of Example 1 were incubated in mouse serum(Cedar Lane) for 15 min and for 30 min at 37° C. Prior to incubation,the lipid associated plasmid was eluted on a DEAE Sepharose CL-6B columnto remove unencapsulated plasmid. Following incubation, an aliquot ofthe incubation mixture was eluted in HBS on a Sepharose CL-4B column.

As a control, 1.5 mg of free ³H-pCMV4-CAT was eluted on a SepharoseCL-4B column in HBS, pH 7.4 (see FIG. 10). For comparison, 1.5 mg offree ³H-pCMV4-CAT was incubated in 500 μL of mouse serum at 37° C. for30 min and eluted in the same manner (see FIG. 11). Note that in FIG.10, the free plasmid eluted in the void volume of the column while, inFIG. 11, the plasmid incubated in serum eluted in the included volumesuggesting that the plasmid had been digested by serum enzymes.

Serum Stability of Plasmid-Lipid Particles Prepared by Reverse Phase

(Particles Prepared from POPC:DODAC:PEG-Cer(C₂₀))

The stability of plasmid-lipid particles was assessed by incubation of a50 μL aliquot in 500 μL of mouse serum (Cedar Lane) for 15 min at 37° C.A 500 μL aliquot of the incubation mixture was eluted in HBS on aSepharose CL-4B column (see FIG. 12). Comigration of the plasmid andlipid in the void volume strongly suggests that no plasmid degradationhas occurred. Any serum associated plasmid or lipid should have beendetected as a peak at around fraction 35 (see control results in FIG.11).

Serum Stability of Plasmid-Lipid Particles Prepared by Dialysis

(Particles Prepared from DOPE:DODAC:PEG-Cer(C₂₀))

A 50 μL aliquot of a particle suspension prepared at a DODAC:plasmidcharge ratio of 4:1 was incubated in 500 μL of mouse serum at 37° C. for30 min and eluted on a Sepharose CL-4B column as described above. FIG.13 shows the elution profile of the sample after incubation in serum. Ascan be seen in FIG. 13, 94% of the plasmid is recovered in the voidvolume suggesting that essentially all of the plasmid recovered fromanion exchange chromatography is encapsulated.

Example 4

This example illustrates the level of plasmid encapsulated in lipidbilayers. Empty lipid complexes containing an aqueous space arerelatively low in density and have a tendency to equilibrate nearer thetop of a density gradient. Free plasmid is relatively high in densityand will therefore equilibrate at a position nearer the bottom of thedensity gradient (where the Ficoll concentration is highest; moredense). Encapsulated plasmid will equilibrate on the gradient at aposition between the positions of the empty lipid complexes and freeplasmid.

Reverse Phase Methods (POPC:DODAC:PEG-Cer(C₂₀))

A 100 μL aliquot of lipid complexes prepared as above but in the absenceof plasmid was added to a Ficoll 400 continuous density gradient(0-7.5%) prepared in HBS (pH 7.4). Similarly a 50 μL aliquot of theplasmid:lipid particle suspension and 0.25 μL of free ³H-pCMV4-CAT wasadded to two separate Ficoll 400 gradients. The samples were centrifugedat 100,000×g for 21 hours. ³H-pCMV4-CAT and ¹⁴C-lipid was assessed in250 μL aliquots in each gradient by scintillation counting.

The empty lipid complexes exhibited a broad range of densities peakingat approximately fraction 25 (see FIG. 14). The broad range was probablydue to heterogeneity in lipid complex or liposome size and lamellaritysince the complexes were only extruded three times through one 200 nmfilter, rather than the usual ten times through two 100 nm filters. Thefree plasmid was present as a single peak near the bottom of thegradient near fraction 35 (see FIG. 15). The gradient profile of theplasmid:lipid particle suspension suggested an association of plasmidwith the lipid as there was comigration of the plasmid and the lipid andthe densities of both were markedly different from that of their freecounterparts (see FIG. 16). The plasmid:lipid ratio was not constantover the gradient profile which can be explained by assuming that notall plasmid-lipid particles contained the same number of plasmidmolecules.

Dialysis Methods (DOPE:DODAC:PEG-Cer(C₂₀))

Plasmid-lipid particles were prepared as described in Example 1 usingdetergent dialysis with a lipid composition of DOPE:DODAC:PEG-Cer(C₂₀)(83.5:6.5:10 mole %). The particles were subjected to density gradientcentrifugation as described for the plasmid-lipid particles prepared byreverse phase methods. The empty lipid complexes were present as asingle peak at about fraction 20 (see FIG. 17). Free plasmid was presentat about fraction 31. It was evident from these controls that successfulentrapment of the pCMV4-CAT was achieved as determined by thecomigration of lipid and plasmid with a peak between that of the freelipid and plasmid controls.

Example 5

This example illustrates the size distribution of plasmid-lipidparticles as measured by quasielastic light scattering using a NicompSubmicron Particle Sizer.

Plasmid-lipid particles were prepared by detergent dialysis as describedin Example 1. The lipid composition was DOPE:DODAC:PEG-Cer(C₂₀)(83.5:6.5:10 mole %). The particles were sized using a Nicomp SubmicronParticle Sizer (see FIG. 18).

The log normal distribution exhibited a x² of 0.2, indicating anextremely homogeneous distribution. The mean diameter of the particleswith entrapped pCMV4-CAT plasmid was 72.4 nm.

Example 6

This example illustrates the clearance and in vivo transfection ofplasmid:lipid particles in mice.

Reverse Phase (POPC:DODAC:PEG-Cer(C₂₀))

Encapsulated plasmid blood clearance was tested in three IRC mice as afunction of percent recovered dose over time. Percent recovery of free³H-plasmid was plotted over a similar time course as a control (see FIG.19). The encapsulated plasmid exhibits a clearance rate which is muchslower than that of the free ³H-plasmid. Additionally, the plasmid:lipidratio does not change significantly over the time course of theexperiment confirming that the plasmid clearance rate is associated withthe clearance rate of the lipid carrier itself.

Detergent Dialysis (DOPE:DODAC:PEG-Cer(C₁₄ or C₂₀))

Fusogenic particles of pCMV4-CAT encapsulated in DOPE:DODAC:PEG-Cer(C₁₄or C₂₀) (83.5:6.5:10 mole %) were prepared as follows:

pCMV4-CAT (50 μg) (42 μL of ³H-pCMV4-CAT; 108 dpm/μL, 1.19 mg/mL) wasincubated with DODAC (407 μg; ˜4:1 DODAC:DNA charge ratio) in 100 μL of1 M OGP and 400 μL of water for 30 min at room temperature. ThisDNA:DODAC complex mixture was added to a suspension of 10 mg ofDOPE:PEG-Cer(C₁₄ or C₂₀) (63.5:10 mole %) and the particles wereconstructed as described in Example 1 (detergent dialysis). Theplasmid-lipid particles for blood clearance studies contained 0.75 μL of¹⁴C-cholesteryl hexadecyl ether (CHE) (6.66 μL/μCi) in 100 μL of 1 M OGPand 400 μL of water. Control particles prepared without DNA contained 2μL of ¹⁴C—CHE for the particles containing PEG-Cer(C₁₄) and 0.75 μL of¹⁴C—CHE for the particles containing PEG-Cer(C₂₀). For in vivotransfection, no ¹⁴C-lipid label was used as it would interfere with theCAT assay.

Clearance of pCMV4-CAT “DNA” Encapsulated in DOPE:DODAC:PEG-Cer (C₁₄ andC₂₀)

External “untrapped” DNA was removed by anion exchange chromatographyusing DEAE Sepharose CL-6B prior to injection into mice. Encapsulationefficiencies were approximately 42% for the systems containingPEG-Cer(C₂₀) and 60% for the systems containing PEG-Cer(C₁₄).

Three groups of three female ICR mice (20-25 g) were injected with 200μL of DNA-encapsulated DOPE:DODAC:PEG-Cer(C₂₀) ((83.5:6.5:10 mole %)each and another set of nine mice were injected with 200 μL ofDNA-encapsulated DOPE:DODAC:PEG-Cer(C₁₄) ((83.5:6.5:10 mole %) each. Onegroup of mice was sacrificed and blood was taken at each of three timepoints (1, 2 and 5 hours). The plasma was separated from whole blood bycentrifugation in 0.5 mL EDTA coated Tainer tubes. A 200 μL aliquot ofthe plasma from each mouse was assayed for ³H-DNA and ¹⁴C-lipid byscintillation counting.

Control particles (no DNA) which had been passed down an anion exchangecolumn also were also analyzed. Two hundred microliters each ofDOPE:DODAC:PEG-Cer(C₂₀) control particles was injected into one group ofthree female ICR mice and 200 μL of DOPE:DODAC:PEG-Cer(C₁₄) controlparticles was injected into three groups of three of the ICR mice. Theplasma was analyzed for ¹⁴C-lipid after 1, 2 and 5 hours.

FIG. 20 shows the clearance of DNA encapsulated in particles composed ofDOPE:DODAC:PEG-Cer(C₂₀) ((83.5:6.5:10 mole %). The DNA and lipid arecleared much less rapidly from the circulation than when PEG-Cer(C₁₄) isused (see FIG. 21). Nearly 50% of the lipid and DNA are present after 1hour. A significant amount of DNA and lipid were still present after 5hr. The amount of DNA and lipid injected was 1.8 μg and 853 μg,respectively. Control particles exhibited a clearance similar to that ofthe plasmid-lipid particles.

FIG. 21 shows the clearance of DNA encapsulated in particles composed ofDOPE:DODAC:PEG-Cer(C₁₄) ((83.5:6.5:10 mole %). Both DNA and lipid arecleared rapidly from the circulation with only about 20% of the lipidand 10% of the DNA present in the plasma after 1 hr. The amount of DNAand lipid injected was 2.7 μg and 912 μg, respectively. Controlparticles exhibited a clearance similar to that of the plasmid-lipidparticles.

In Vivo Transfection in Lung, Liver and Spleen

Three groups of four IRC mice were injected via tail vein with pCMV4-CATencapsulated in lipid particles composed of DOPE:DODAC:PEG-Cer(C₁₄)(83.5:6.5:10 mole %, “A”) or DOPE:DODAC:PEG-Cer(C₂₀) (83.5:6.5:10 mole%, “B”), prepared as described above. The mice were sacrificed after 2,4 and 8 days and the lung, liver and spleen were assayed for CATactivity according to a modification of Deigh, Anal. Biochem.156:251-256 (1986). The amount of plasmid injected was 2.6 μg for theparticles containing PEG-Cer(C₁₄) and 1.5 μg for the particlescontaining PEG-Cer(C₂₀).

FIG. 22 shows the results of in vivo transfection achieved in the lung.As can be seen from this figure, treatment with formulation “A” providedexcellent transfection efficiency (based on CAT activity) up to 4 days.Formulation “B”, while resulting in overall lower levels of CATactivity, provided relatively constant levels of enzyme activity over 8days.

FIG. 23 shows the results of transfection achieved in the liver. Forboth formulations, transfection (and CAT activity) reached a maximum at4 days.

FIG. 24 shows the results of transfection achieved in the spleen whereinthe maximum transfection was found for both formulations to occur after2 days.

VII. CONCLUSION

As discussed above, in accordance with one of its aspects, the presentinvention provides methods for preparing serum-stable plasmid-lipidparticles which are useful for the transfection of cells, both in vitroand in vivo.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated by reference into thespecification to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto be incorporated herein by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent applications, foreign patents, foreign patentapplications and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet, including butnot limited to U.S. patent application Ser. No. 10/956,425 and U.S. Pat.Nos. 6,815,432, 6,534,484, and 5,981,501, are incorporated herein byreference, in their entirety. Aspects of the embodiments can bemodified, if necessary to employ concepts of the various patents,applications and publications to provide yet further embodiments. Theseand other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

1. A method for the preparation of serum-stable plasmid-lipid particles,comprising: (a) combining a plasmid with cationic lipids in a detergentsolution to provide a coated plasmid-lipid complex; (b) contactingnon-cationic lipids with said coated plasmid-lipid complex to provide asolution comprising detergent, a plasmid-lipid complex and non-cationiclipids; and (c) removing said detergent from said solution of step (b)to provide a solution of serum-stable plasmid-lipid particles, whereinsaid plasmid is encapsulated in a lipid bilayer and said particles areserum-stable and have a size of from about 50 to about 150 nm.
 2. Amethod in accordance with claim 1, wherein said removing is by dialysis.3. A method in accordance with claim 1, wherein step (b) furthercomprises adding a polyethylene glycol-lipid conjugate.
 4. A method inaccordance with claim 2, 3, wherein said polyethylene glycol-lipidconjugate is a PEG-ceramide conjugate.
 5. A method in accordance withclaim 1, further comprising; (d) sizing said particles to achieve auniform particle size.
 6. A method in accordance with claim 1, whereinsaid cationic lipids are selected from the group consisting of DODAC,DDAB, DOTAP, DOTMA, DOSPA, DOGS, DC-Chol and combinations thereof.
 7. Amethod in accordance with claim 1, wherein said non-cationic lipids areselected from the group consisting of DOPE, POPC, EPC and combinationsthereof.
 8. A method in accordance with claim 1, wherein said detergentsolution comprises a detergent having a critical micelle concentrationof between about 20 mM and 50 mM.
 9. A method in accordance with claim8, wherein said detergent is n-octyl-β-D-glucopyranoside.
 10. A methodfor the preparation of serum-stable plasmid-lipid particles, comprising;(a) preparing a mixture comprising cationic lipids and non-cationiclipids in an organic solvent; (b) contacting an aqueous solution ofplasmid with said mixture prepared in step (a) to provide a clear singlephase; and (c) removing said organic solvent to provide a suspension ofplasmid-lipid particles, wherein said plasmid is encapsulated in a lipidbilayer, and said particles are stable in serum and have a size of fromabout 50 to about 150 nm.
 11. A method in accordance with claim 10,wherein said non-cationic lipids comprise a polyethylene glycol-lipidconjugate.
 12. A method in accordance with claim 11, wherein saidpolyethylene glycol-lipid conjugate is a PEG-ceramide conjugate.
 13. Amethod in accordance with claim 10, further comprising; (d) sizing saidplasmid-lipid particles to achieve a uniform particle size.
 14. A methodin accordance with claim 10, wherein said cationic lipids are selectedfrom the group consisting of DODAC, DDAB, DOTAP, DOTMA, DOSPA, DOGS,DC-Chol and combinations thereof.
 15. A method in accordance with claim10, wherein said non-cationic lipids are selected from the groupconsisting of DOPE, POPC, EPC and combinations thereof.
 16. Aplasmid-lipid particle prepared according to claim
 1. 17. A method forintroducing a plasmid into a cell, comprising; (a) preparing aplasmid-lipid particle according to the method of claim 1; and (b)contacting said cell with said plasmid-lipid particle for a period oftime sufficient to introduce said plasmid into said cell.
 18. A methodin accordance with claim 17, wherein said plasmid-lipid particlecomprises a plasmid, DODAC, POPC and a PEG-Ceramide selected from thegroup consisting of PEG-Cer-C₂₀ and PEG-Cer-C₁₄.
 19. A method inaccordance with claim 17, wherein said plasmid-lipid particle comprisesa plasmid, DODAC, DOPE and a PEG-Ceramide selected from the groupconsisting of PEG-Cer-C₂₀ and PEG-Cer-C₁₄.
 20. A plasmid-lipid particleprepared according to claim
 10. 21. A method for introducing a plasmidinto a cell, comprising; (a) preparing a plasmid-lipid particleaccording to the method of claim 10; and (b) contacting said cell withsaid plasmid-lipid particle for a period of time sufficient to introducesaid plasmid into said cell.
 22. A method in accordance with claim 21,wherein said plasmid-lipid particle comprises a plasmid, DODAC, POPC anda PEG-Ceramide selected from the group consisting of PEG-Cer-C₂₀ andPEG-Cer-C₁₄.
 23. A method in accordance with claim 21, wherein saidplasmid-lipid particle comprises a plasmid, DODAC, DOPE and aPEG-Ceramide selected from the group consisting of PEG-Cer-C₂₀ andPEG-Cer-C₁₄.